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Research Paper
J Vasc Res 2017;54:344–358
DOI: 10.1159/000478984
Received: June 15, 2017
Accepted: June 21, 2017
Published online: October 25, 2017
Low-Frequency Components in Rat Pial
Arteriolar Rhythmic Diameter Changes
Dominga Lapi a Teresa Mastantuono a Martina Di Maro a Maurizio Varanini b
Antonio Colantuoni a
a
Department of Clinical Medicine and Surgery, Federico II University Medical School, Naples, and b CNR Institute of
Clinical Physiology, Pisa, Italy
Abstract
This study aimed to analyze the frequency components
present in spontaneous rhythmic diameter changes in rat
pial arterioles. Pial microcirculation was visualized by fluorescence microscopy. Rhythmic luminal variations were
evaluated via computer-assisted methods. Spectral analysis
was carried out on 30-min recordings under baseline conditions and after administration of acetylcholine (Ach), papaverine (Pap), Nω-nitro-L-arginine (L-NNA) prior to Ach, indomethacin (INDO), INDO prior to Ach, charybdotoxin and apamin, and charybdotoxin and apamin prior to Ach. Under
baseline conditions all arteriolar orders showed 3 frequency
components in the ranges of 0.0095–0.02, 0.02–0.06, and
0.06–0.2 Hz, another 2 in the ranges of 0.2–2.0 and 2.5–4.5
Hz, and another ultra-low-frequency component in the
range of 0.001–0.0095 Hz. Ach caused a significant increase
in the spectral density of the frequency components in the
range of 0.001–0.2 Hz. Pap was able to slightly increase spectral density in the ranges of 0.001–0.0095 and 0.0095–0.02
© 2017 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/jvr
Hz. L-NNA mainly attenuated arteriolar responses to Ach.
INDO prior to Ach did not affect the endothelial response to
Ach. Charybdotoxin and apamin, suggested as endothelium-derived hyperpolarizing factor inhibitors, reduced spectral density in the range of 0.001–0.0095 Hz before and after
Ach administration. In conclusion, regulation of the blood
flow distribution is due to several mechanisms, one of which
is affected by charibdotoxin and apamin, modulating the
vascular tone.
© 2017 S. Karger AG, Basel
Introduction
Control of the blood flow in the peripheral tissues is
crucial to maintaining the complex functions of body organs, requiring appropriate modulation of blood supply
to capillary networks. The main vessels designed to regulate the blood flow supply to the tissues are arterioles, and
they are able to contract and dilate, causing rhythmic diameter changes known as vasomotion [1, 2]. This activity
has been intensively studied under in vivo and in vitro
conditions utilizing different experimental models [3]. It
has been suggested that the rhythmic diameter changes
are mainly due to the activity of vascular smooth muscle
Dr. Dominga Lapi
Department of Clinical Medicine and Surgery
Federico II University Medical School, Via S. Pansini 5
IT–80121 Naples (Italy)
E-Mail d.lapi @ dfb.unipi.it
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Keywords
Rat pial arterioles · Vasomotion · Low-frequency oscillations ·
Spectral analysis · Nitric oxide · Endothelium-derived
hyperpolarizing factor · Prostaglandins
Cerebral Vasomotion and LF Oscillations
changes in rat pial arterioles in order to define their frequency components. The main aim was to test the arteriolar responses to the following substances able to dilate
or inhibit the dilation of vessels: acetylcholine (Ach),
known to facilitate endothelium-dependent arteriolar dilation; Nω-nitro-L-arginine (L-NNA), inhibitor of eNOS;
indomethacin (INDO), inhibitor of prostaglandin endoperoxidase; and charybdotoxin plus apamin, inhibitors of
EDHF [15]. Previous studies have indicated that dilation
of pial vessels is resistant to inhibitors of NO synthase and
cyclooxygenase, suggesting a role for a third endothelium-dependent mechanism in cerebral vessel tone modulation and blood flow distribution [16–21]. Consequently, we tested arteriolar responses to papaverine (Pap),
known as an NO-independent vasodilator. Thirty-minute long-term tracings of arteriolar rhythmic diameter
changes were analyzed to obtain the resolution of the VLF
components according to previous results in humans
[22].
Methods
Experimental Groups
This study was carried out on male Wistar rats (250–300 g)
randomly assigned to the following 8 groups: (1) a sham-operated
group (S, n = 11), subjected to the same surgical procedures as the
experimental groups but without drug treatment; (2) an Ac group
(n = 11), administered a topical application of Ach (10 μM) [23];
(3) a Pap group (n = 11), treated with a topical application of Pap
(0.1 mM), a nitric oxide-independent vasodilator [24]; (4) an LAc
group (n = 11), treated with L-NNA (100 μM) prior to Ach (10 μM),
topically applied [25]; (5) an I group (n = 11), administered a topical application of INDO (10 μM) [26]; (6) an IA group (n = 11),
administered topical treatment of INDO (10 μM) prior to Ach (10
μM); (7) a CAp group (n = 11), administered charybdotoxin (100
nM) plus apamin (10 nM) topically [27]; and (8) a CApAc group
(n = 11), treated with charybdotoxin (100 nM) plus apamin (10 nM)
prior to Ach (10 μM) topically administered.
The drugs were topically administered during the whole 30min observation period, and they were purchased from Sigma
Chemical (St. Louis, MO, USA).
Animal Preparation
Anesthesia was induced with α-chloralose (50 mg/kg of body
weight, i.p.) and maintained with supplemental α-chloralose (30
mg/kg of body weight, i.v. every h). Rats were tracheotomized, paralyzed with tubocurarine chloride (1 mg/kg × h, i.v.) and mechanically ventilated with room air and supplemental oxygen. Catheters were placed in the left femoral artery for arterial blood pressure recording and blood gases sampling and in the right femoral
vein for injection of the fluorescent tracer, i.e., fluorescein isothiocyanate bound to dextran (molecular weight: 70 kDa [FD 70], 50
mg/100 g of body weight, i.v. as a 5% weight/volume solution), for
3 min administered once at the beginning of the experiment. Blood
gas measurements were carried out on arterial blood samples with-
J Vasc Res 2017;54:344–358
DOI: 10.1159/000478984
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cells [4–6]. Moreover, the oscillations of vessel diameters
are related to a combination of endothelium- and nonendothelium-dependent factors. Several studies have been
carried out to assess the components of this vasomotor
activity and the molecules that affect arteriolar diameter
changes [7].
Previous studies carried out on human skin blood flow
oscillations, recorded by laser Doppler perfusion monitoring (LDPM), have indicated that variations in blood
flow are related to specific factors. The LDPM recordings,
derived from human skin, were analyzed via different
methods, such as Fourier and wavelet transforms [8–11].
These techniques demonstrated that several frequency
components could be identified entrapped in the range of
0.0095–2.0 Hz (0.57–120 cycles per min [cpm]). After
many efforts, 5 main components were identified in cutaneous blood flow oscillations according to the frequency ranges: low- (LF) and high-frequency (HF) components. The LF one was related to myogenic activity, i.e.,
contraction and relaxation of vascular smooth muscle
cells, endowed in the wall of arterioles, in the frequency
range of 0.06–0.2 Hz (3.6–12 cpm). Another frequency
component (0.02–0.06 Hz/1.2–3.6 cpm) was linked to the
sympathetic nervous system discharge on the arteriolar
vessels, usually on the larger arterioles (average diameter
<50 μm). The lowest-frequency component was correlated to the release of nitric oxide (NO) from endothelial
cells, causing oscillations of arteriolar diameters in the
range of 0.0095–0.02 Hz (0.57–1.2 cpm). Moreover, 2
higher-frequency components were suggested in the
ranges of 0.2–0.4 Hz (12–24 cpm) and 0.4–1.5 Hz (24–90
cpm), correlated to respiration and heart rate, respectively. In a further study analyzing very long tracings derived
from human skin (30-min recordings), a sixth component was described, independently of NO release (0.001–
0.0095 Hz/0.06–0.57 cpm), and defined a very low-frequency (VLF) component [12]. We tried to assess whether the oscillations in vessel diameters, visualized by direct
observation of arterioles in an experimental model, presented frequency components in ranges previously reported in humans. We chose the rat cranial window preparation implemented on the left parietal cortex [13, 14],
where we could record the rhythmic diameter changes of
rat pial arterioles by intravital fluorescence microscopy,
hypothesizing that the arteriolar NO-independent component could be evaluated after inhibition of the suggested endothelium-derived hyperpolarizing factor (EDHF)
by charybdotoxin and apamin.
Therefore, the purpose of the present study was to detect and analyze the vivo spontaneous rhythmic diameter
drawn from the arterial catheter at 30-min intervals (ABL5 radiometer; Copenhagen, Denmark). Mean arterial blood pressure,
heart rate, respiratory CO2, and blood gas values were recorded
and remained stable within the physiological ranges. Rectal temperature was monitored and preserved at 37.0 ± 0.5 ° C with a heating stereotaxic frame where the rats were secured.
To observe the pial microcirculation, a closed cranial window
(4 × 5 mm) was implanted above the left parietal cortex (posterior,
1.5 mm to the bregma; lateral, 3 mm to the midline), as previously
described [28].
All experiments conformed to the Guide for the Care and Use
of Laboratory Animals published by the US National Institutes of
Health (NIH Publication No. 85-23, revised 1996) and to institutional rules for the care and handling of experimental animals. The
protocol was approved by the Federico II University of Naples Ethical Committee.
J Vasc Res 2017;54:344–358
DOI: 10.1159/000478984
Order
Arterioles, n
Diameter, μm
Length, μm
Rats, n
5
4
3
2
1
28
85
108
120
176
62.8 ± 2.3*
43.7 ± 1.4*
33.5 ± 1.2*
23.8 ± 1.0*
16.4 ± 0.8*
1,114 ± 23
885.4 ± 15.3
473 ± 10
364.0 ± 7.5
155.0± 5.8
88
88
88
88
88
Intravital Microscopy Technique and Microvascular
Parameter Evaluation
Observations of pial vessels were conducted using a fluorescence microscope (Leitz Orthoplan) fitted with long-distance objectives (×2.5, numerical aperture [NA] 0.08; ×10, NA 0.20; ×20,
NA 0.25; and ×32, NA 0.40), a ×10 eyepiece, and a filter block (Ploemopak; Leitz). Epi-illumination was provided by a 100-W mercury lamp using the appropriate filters for FITC and a heat filter
(KG1; Leitz). The pial microcirculation was televised with a DAGE
MTI 300RC low-light level digital camera and recorded by a computer-based frame grabber (Pinnacle DC 10plus; Avid Technology, MA, USA).
Microvascular measurements were made offline using a computer-assisted imaging software system (MIP Image; CNR, Institute of Clinical Physiology, Pisa, Italy). Visualization of the pial
microcirculation was performed for 10 min under baseline conditions after 2 min of FITC administration and then for 30 min continuously.
Arteriolar diameters were measured via a computer-assisted
method (MIP Image, frame by frame). To avoid bias due to singleoperator measurements, 2 independent “blinded” operators measured the vessel diameters. Their measurements overlapped in all
cases. The measurements were compared with those obtained via
the automated method (MIP Automated Image program), where
the vessel walls were automatically identified. These measurements overlapped in all cases. Moreover, the measurements were
compared with those obtained via the shearing method (±0.5 μm).
Under baseline conditions, the arteriolar network was mapped
by stop frame images and pial arterioles were classified according
to a centripetal ordering scheme (Strahler’s method, modified according to diameter), as previously described [29, 30]. Order 0 was
assigned to the capillaries; thereafter, the terminal arterioles were
assigned order 1 and the vessels upstream were assigned progressively higher orders.
Mean arterial blood pressure (Viggo-Spectramed P10E2 transducer; Oxnard, CA; connected to a catheter in the femoral artery)
and heart rate were monitored with a Gould Windograf recorder
(model 13-6615-10S; Gould, OH, USA). Data were recorded and
stored in a computer.
The rhythmic variations in the diameter of pial arterioles were
analyzed via a computer-assisted power spectrum method, such as
the generalized short-time Fourier transform [31–33], a multiresolution transform which allows one to choose, at each frequency,
346
line conditions
* p < 0.01 vs. a different order.
the most appropriate balance between time and frequency resolution according to the user’s requirements. A Hamming window
was used and spectra were computed at frequencies spaced proportionally to the frequency resolution. The power density spectral
distribution was obtained by time averaging the time-frequency
power density representation. This technique permits evaluation
of nonstationary data, such as those represented by rhythmic variations in vessel diameters [34].
Statistical Analysis
All data are expressed as means ± SEM. Data were tested for a
normal distribution with the Kolmogorov-Smirnov test. Parametric (Student’s t tests, ANOVA and Bonferroni’s post hoc test) or
nonparametric tests (Wilcoxon, Mann-Whitney, and KruskalWallis tests) were used; nonparametric tests were applied to compare diameter and length data among the experimental groups.
The statistical analysis was carried out using the SPSS 14.0 statistical package. p < 0.05 was considered statistically significant.
Results
Arterioles were classified according to a centripetal ordering scheme (Strahler’s method, modified according to
diameter) in 5 orders according to diameter, length, and
branchings, as previously reported [30]. Capillaries, assigned order 0, originated from the smallest arterioles
(order 1, average maximum diameter 16.4 ± 0.8 μm and
average length 155.0 ± 5.8 μm). The largest pial arterioles,
i.e., order 5, showed an average maximum diameter of
62.8 ± 2.3 μm and a length of 1,114 ± 23 μm. The values
of diameters and lengths were significantly different
among the arteriolar orders (p < 0.01, Kruskal-Wallis test;
Table 1).
Under baseline conditions in all preparations (n = 88),
all pial arterioles (n = 517) exhibited rhythmic diameter
changes. In Figures 1–3 we report the changes in arteriolar diameter of one order 2, one order 3 and one order 4
arteriole, respectively.
Lapi/Mastantuono/Di Maro/Varanini/
Colantuoni
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Table 1. Diameter and length of each arteriolar order under base-
Color version available online
Diameter, μm
27
26
25
24
23
22
PSD, μm2/component
a
0
5
10
15
Time, min
20
25
30
0.15
0.10
0.05
0
1
b
10
Frequency, cpm
100
200
300
100
90
pressed as percent normalized power spectral density (PSD: μm2/Hz) in sham-operated rats. cpm, cycles per minute; ULF,
ultra-low frequency; VLF, very low frequency; ILF, intermediate frequency; LF,
low frequency; HF, high frequency; VHF,
very high frequency (n = 11 order 2 arterioles).
70
60
50
40
30
20
10
c
0
ULF
VLF
ILF
LF
HF
VHF
The average minimum and maximum diameters of
arterioles are reported in Table 2. It is possible to observe that oscillations in arteriolar diameter (amplitude: % changes in mean diameter) were 21.5 ± 2.5% in
order 2, 22.4 ± 3.2% in order 3, and 5.7 ± 1.6% in order
4 vessels.
Topical application of Ach caused an increase in the
average maximum and minimum arteriolar diameters,
with a consequent reduction in oscillation amplitude.
The average maximum diameter of order 2 arterioles
(n = 11) was 33.2 ± 1.2 versus 24.7 ± 1.4 μm (baseline),
while the average mean diameter was 32.6 ± 0.9 versus
22.5 ± 1.3 μm (baseline). Therefore, the average mean diameter increased by 44.9 ± 3.5% in relation to baseline
(p < 0.01 vs. baseline, Wilcoxon’s test). The amplitude of
oscillations was reduced (5.1 ± 0.7% of the mean diameter) compared to baseline.
Pap topically applied induced a marked dilation of order 2 arterioles; the average maximum diameter of order
2 arterioles (n = 11) was 34.1 ± 1.8 versus 25.3 ± 1.6 μm
(baseline), while the average mean diameter was 33.0 ±
1.1 versus 23.8 ± 1.0 μm (baseline). Therefore, the average
mean diameter increased by 45.7 ± 2.3% in relation to
baseline (p < 0.01 vs. baseline, Wilcoxon/Mann-Whitney
Cerebral Vasomotion and LF Oscillations
J Vasc Res 2017;54:344–358
DOI: 10.1159/000478984
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Fig. 1. Rhythmic diameter changes in an
order 2 arteriole (a) and the main corresponding frequency components (b, c), ex-
Normalized PSD, %
80
Color version available online
Diameter, μm
35.5
35.0
34.5
34.0
33.5
33.0
PSD, μm2/cpm
a
0
5
10
15
Time, min
25
20
30
0.015
0.010
0.005
0
1
b
10
Frequency, cpm
100
200
300
100
90
pressed as percent normalized power spectral density (PSD: μm2/Hz) in sham-operated rats. cpm, cycles per minute; ULF,
ultra-low frequency; VLF, very low frequency; ILF, intermediate frequency; LF,
low frequency; HF, high frequency; VHF,
very high frequency (n = 11 order 3 arterioles).
J Vasc Res 2017;54:344–358
DOI: 10.1159/000478984
60
50
40
30
20
10
c
tests). The amplitude of oscillations was reduced (7.2 ±
0.4% of the mean diameter) compared to baseline.
Inhibition of eNOS by L-NNA prior to Ach abolished
the dilation induced by Ach. The average maximum diameter of order 2 arterioles (n = 11) was not significantly
different compared to baseline (24.8 ± 1.3 vs. 25.3 ± 1.1
μm, baseline). Consequently, the average mean diameter
did not significantly change.
Indomethacin, an inhibitor of cyclooxygenase, did
not significantly affect the diameter of order 2 arterioles
(n = 11); the average maximum diameter was 25.9 ± 1.4
versus 26.1 ± 1.5 μm (baseline). Therefore, there were no
348
70
0
ULF
VLF
ILF
LF
HF
VHF
significant changes in any of the microvascular parameters.
Administration of INDO prior to Ach did not significantly affect the dilation induced by Ach. The average
mean diameter of order 2 arterioles (n = 11) was 32.2 ±
1.3 versus 23.5 ± 0.9 μm (baseline) (p < 0.01 vs. baseline,
Wilcoxon’s test); consequently, the percent increase in
mean diameter after INDO plus Ach was 37.0 ± 2.5 in relation to baseline.
Charybdotoxin plus apamin significantly decreased
the average mean diameter of order 2 arterioles (n = 11),
i.e., 17.5 ± 0.8 versus 22.9 ± 1.2 μm (baseline) (p < 0.01 vs.
Lapi/Mastantuono/Di Maro/Varanini/
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Fig. 2. Rhythmic diameter changes in an
order 3 arteriole (a) and the main corresponding frequency components (b, c), ex-
Normalized PSD, %
80
Color version available online
Diameter, μm
43.5
43.0
42.5
42.0
41.5
41.0
a
0
5
10
15
Time, min
20
25
30
0.010
0.005
b
Fig. 3. Rhythmic diameter changes in an
order 4 arteriole (a) and the main corresponding frequency components (b, c), ex-
pressed as percent normalized power spectral density (PSD: μm2/Hz) in sham-operated rats. cpm, cycles per minute; ULF,
ultra-low frequency; VLF, very low frequency; ILF, intermediate frequency; LF,
low frequency; HF, high frequency; VHF,
very high frequency (n = 11 order 4 arterioles). In order 4 arterioles ULF and VLF
prevailed compared to observations in order 2 and 3 arterioles. * p < 0.01 vs. order 2
and 3 arterioles.
1
10
Frequency, cpm
100
200
300
100
90
80
70
60
50
40
30
20
10
c
0
*
*
ULF
VLF
ILF
LF
HF
VHF
baseline, Wilcoxon’s test); the percent decrease was 26.2
± 2.5% in relation to baseline. Oscillations in mean diameter were reduced after charybdotoxin plus apamin administration, i.e., up to 9.9 ± 0.8%, which is significantly
different compared to 24.1 ± 1.2% under baseline conditions (Wilcoxon’s test).
Charybdotoxin plus apamin administration prior to
Ach injection diminished the dilation of order 2 arterioles
(n = 11); the average mean diameter was 28.0 ± 0.7 versus
22.0 ± 1.2 μm (baseline) (p < 0.01 vs. baseline, Wilcoxon’s
test). The increase in the average mean diameter induced
by charybdotoxin plus apamin plus Ach was 27.2 ± 1.5%
in relation to baseline. After charybdotoxin plus apamin
plus Ach, oscillations in mean diameter were reduced,
i.e., up to 5.8 ± 0.8%, compared to 25.8 ± 1.2% under baseline conditions.
Power spectrum analysis of rhythmic diameter changes was carried out on 30-min recordings via a generalized
short-time Fourier transform. Previous data indicated
that 30-min recordings were sufficient to achieve good
resolution of the VLF components [22]. Our data indicate
that several frequency components were involved in the
luminal oscillations. The first component was in the range
of 0.001–0.0095 Hz (0.06–0.57 cpm, ultra-low-frequency [ULF] component); the second was in the range of
0.0095–0.02 Hz (0.57–1.2 cpm, VLF component); the
Cerebral Vasomotion and LF Oscillations
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0
Normalized PSD, %
PSD, μm2/cpm
0.015
350
J Vasc Res 2017;54:344–358
DOI: 10.1159/000478984
Table 2. Minimum, maximum, and mean diameters (average) of
order 2 arterioles in the different experimental groups under baseline conditions and after treatment
Group
Average
minimum
diameter,
μm
Average
maximum
diameter,
μm
Average
mean
diameter,
μm
Arterioles,
n
Ac
20.5 ± 1.0
31.5 ± 1.3*
22.5 ± 1.3
31.8 ± 1.5*
21.5 ± 1.2
20.7 ± 1.0
20.5 ± 1.2
21.1 ± 1.3
21.1 ± 1.2
30.2 ± 1.0*
20.3 ± 1.0
16.4 ± 1.2*
19.2 ± 0.7
27.1 ± 0.9 *
24.7 ± 1.4
33.2 ± 1.2*
25.3 ± 1.6
34.1 ± 1.8*
25.3 ± 1.1
24.8 ± 1.3
26.1 ± 1.5
25.9 ± 1.4
25.4 ± 1.4
34.5 ± 1.5*
25.9 ± 1.4
18.1 ± 0.9*
24.9 ± 0.9
29.0 ± 1.2*
22.5 ± 1.3
32.6 ± 0.9*
23.8 ± 1.0
33.0 ± 1.1*
23.1 ± 1.5
22.4 ± 1.2
23.8 ± 1.2
24.0 ± 0.6
23.5 ± 0.9
32.2 ± 1.3*
22.9 ± 1.2
17.5 ± 0.8*
22.0 ± 1.2
28.0 ± 0.7*
11
B
T
Pap
B
T
LAc
B
T
I
B
T
IA
B
T
CAp
B
T
CApAc B
T
11
11
11
11
11
11
B, baseline; T, after treatment; Ac, administered a topical application of acetylcholine (10 μM); Pap, treated with a topical application of papaverine (0.1 mM); LAc, treated with Nω-nitro-Larginine (100 μM) prior to acetylcholine (10 μM), topically applied;
I, administered a topical application of indomethacin (10 μM); IA,
administered topical treatment of indomethacin (10 μM) prior to
acetylcholine (10 μM); Cap, administered charybdotoxin (100 nM)
plus apamin (10 nM) topically; CApAc, treated with charybdotoxin (100 nM) plus apamin (10 nM) prior to acetylcholine (10 μM)
topically administered. * p < 0.01 vs. baseline conditions.
sity of the ILF and LF components was not statistically
significant (11.5 ± 1.3 vs. 13.3 ± 1.5% and 22.9 ± 2.1 vs.
26.0 ± 1.9%, respectively; p = ns). Moreover, Pap caused
an increase in power spectrum oscillations compared to
those observed after Ach administration (Fig. 5).
Topical application of L-NNA, an inhibitor of nitric
oxide synthase, prior to Ach administration, blunted the
order 2 arteriole responses to Ach. The total power spectral density of diameter oscillations, however, increased
compared to baseline and the values of sham-operated
animals (0.470 ± 0.015 vs. 0.385 ± 0.018 μm2/Hz, baseline;
p < 0.01, ANOVA). It is worth noting that the percent
spectral density of the ULF and VLF components dramatically decreased compared to Ach-induced oscillations (5.0 ± 0.9 vs. 29.3 ± 2.5%, p < 0.01, ANOVA; 1.0 ±
0.2 vs. 23 ± 2%, p < 0.01, ANOVA; and p < 0.05, Bonferroni’s post hoc test; respectively). In particular, the effects
of L-NNA prior to Ach significantly affected the VLF
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third was in the range of 0.02–0.06 Hz (1.2–3.6 cpm, intermediate low-frequency (ILF) component); the fourth
was in the range of 0.06–0.2 Hz (3.6–12 cpm, LF component); the fifth was in the range of 0.2–2.0 Hz (12–120
cpm, HF component), and the sixth was in the range of
2.0–4.5 Hz (120–270 cpm, very high-frequency [VHF]
component). The VHF component presented the highest-power spectral density in order 2 arterioles (n = 120),
followed by the LF, ILF, ULF, and VLF components.
In order 3 arterioles (n = 108), the same distribution of
spectral density was observed, while in order 4 vessels
(n = 85) the VHF component presented the highest spectral density, but there was an increase in ULF and VLF
with a concomitant decrease in ILF and HF spectral density (Fig. 2, 3).
To clarify the factors involved in these oscillatory components, we tested the effects of several substances on the
arteriolar diameter changes in order 2 arterioles. Topical
application of Ach caused an increase in the total spectral
density of diameter oscillations (0.520 ± 0.015 vs. 0.348 ±
0.019 μm2/Hz, baseline; p < 0.01, ANOVA), and the values were significantly different compared to baseline and
the values of sham-operated animals [35]. Therefore, application of Ach induced an increase in the power of oscillations. Moreover, taking into account the relative
power spectral density of each component, ULF and VLF
ones revealed a percent increase in spectral density compared to baseline and those evaluated in sham-operated
animals (29.3 ± 2.5 vs. 5.2 ± 0.5% and 23 ± 2 vs. 4.1 ± 0.8%,
respectively; p < 0.01, ANOVA). HF and VHF components decreased in their percent power (2.0 ± 0.5 vs. 9.7 ±
0.9% and 3.0 ± 1.1 vs. 41.0 ± 2.4%, respectively; p < 0.01,
ANOVA), while ILF and LF components did not significantly change, even though the trend was toward an increase for ILF and a decrease for LF (Fig. 4).
Pap induced an increase in the total spectral density of
diameter oscillations (0.610 ± 0.018 vs. 0.370 ± 0.020 μm2/
Hz, baseline; p < 0.01, ANOVA), anf the values were significantly different compared to baseline and the values
of sham-operated animal. Moreover, an increase was detected in the percent spectral density of the ULF and VLF
components compared to baseline and the values of
sham-operated animals (13.7 ± 1.2 vs. 5.2 ± 0.5% and 13.5
± 1.6 vs. 4.1 ± 0.8%, respectively; p < 0.01, ANOVA, and
p < 0.05, Bonferroni’s post hoc test). The HF and VHF
components slightly decreased in their percent power
compared to baseline and the values of sham-operated
rats (6.2 ± 0.8 vs. 9.7 ± 1.5% and 32.2 ± 3.5 vs. 41 ± 2%,
respectively; p < 0.01, ANOVA, and p < 0.05, Bonferroni’s
post hoc test). The decrease in the percent spectral den-
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33
Diameter, μm
32
31
30
29
28
a
0
5
10
15
Time, min
20
25
30
pressed as percent normalized power spectral density (PSD: μm2/Hz) in a rat treated
with acetylcholine. cpm, cycles per minute.
Acetylcholine caused an increase in ULF
and VLF spectral density. p < 0.01 vs. baseline conditions.
0.10
0.05
0
b
1
10
Frequency, cpm
100
200
300
component, which was completely abolished; the ULF
component was reduced but not abolished.
The ILF component did not significantly change (17.0
± 1.2 vs. 18.7 ± 1.3%), while the relative spectral density
of the LF, HF, and VHF components presented an increase compared to the Ach-triggered oscillatory components (31.0 ± 2.5 vs. 24.0 ± 1.7, p < 0.01, ANOVA, and
p < 0.05, Bonferroni’s post hoc test; 10 ± 1 vs. 2.0 ± 0.5%,
p < 0.01, ANOVA; and 36.0 ± 2.3 vs. 3.0 ± 1.1%, p < 0.01,
ANOVA, and p < 0.05, Bonferroni’s post hoc test, respectively; Fig. 6).
INDO (COX 1 inhibitor) administration did not significantly affect the spectral density of all of the frequency
components. INDO applied prior to Ach on pial arterioles did not influence the power spectral density of the
ULF and VLF components; indeed, these did not change
compared to the values detected after Ach local administration.
Topical administration of charybdotoxin plus apamin, suggested to inhibit the endothelium-derived hyperpolarizing factor, caused an increase in the total spectral density of diameter oscillations (0.420 ± 0.020 vs.
0.355 ± 0.022 μm2/Hz, baseline; p < 0.01, ANOVA),
which were significantly different compared to baseline
and the values of sham-operated animals [36]. Charybdotoxin plus apamin abolished the ULF component,
while the VLF one was not affected. The relative power
spectral density of each component indicates that the
percent spectral density of the ULF component significantly decreased (0.3 ± 0.1 vs. 5.2 ± 0.5%, p < 0.01, ANOVA, and p < 0.05, Bonferroni’s post hoc test) compared
to baseline and the values of sham-operated animals. The
VLF component did not significantly change (4.4 ± 0.7
vs. 4.1 ± 0.8%), while the percent spectral density of the
ILF and LF components significantly increased compared to baseline and the values of sham-operated animals (19.6 ± 1.0 vs. 14.0 ± 0.9%, p < 0.01, ANOVA, and
45.3 ± 1.8 vs. 26 ± 2%, p < 0.01, ANOVA, and p < 0.05,
Bonferroni’s post hoc test, respectively). The HF and
VHF components decreased in terms of their percent
power (5.4 ± 1.0 vs. 9.7 ± 0.9% and 25.0 ± 1.6 vs. 41.0 ±
2.4%, respectively; p < 0.01, ANOVA, and p < 0.05, Bonferroni’s post hoc test; Fig. 7).
Charybdotoxin plus apamin, administrated before
Ach injection, induced an increase in the total spectral
density of diameter oscillations (0.540 ± 0.025 vs. 0.345 ±
0.015 μm2/Hz, baseline; p < 0.01, ANOVA), and the values were significantly different compared to baseline and
Cerebral Vasomotion and LF Oscillations
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Fig. 4. Rhythmic diameter changes in an
order 2 arteriole (a) and the main corresponding frequency components (b), ex-
PSD, μm2/cpm
0.15
Color version available online
36
Diameter, μm
35
34
33
32
31
a
0
5
10
15
Time, min
20
30
PSD, μm2/cpm
0.15
0.10
0.05
0
b
1
10
Frequency, cpm
100
200
300
Fig. 5. Rhythmic diameter changes in an order 2 arteriole (a) and the main corresponding frequency components
(b), expressed as percent normalized power spectral density (PSD: μm2/Hz) in a rat treated with papaverine. cpm,
cycles per minute. Papaverine caused a decrease in ULF and VLF spectral density. ◇p < 0.01 vs. the acetylcholinetreated group.
352
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Topical application of all of the substances did not affect heart (240 ± 30 beats per minute) or respiratory rates
(60 ± 20 breaths per min).
Discussion
The data of the present study indicate that pial arterioles exhibit rhythmic diameter changes characterized by
several frequency components, identified by power spectrum analysis. In our experiments the lowest-frequency
component was detected in the range of 0.001–0.0095 Hz
(0.06–0.57 cpm, ULF component). The other LF components were identified in the ranges of 0.0095–0.02 Hz
(0.57–1.2 cpm, VLF component), 0.02–0.06 Hz (1.2–3.6
cpm, ILF component), and 0.06–0.2 Hz (3.6–12 cpm, LF
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the values of sham-operated animals [35]. The relative
power spectral density of the ULF and VLF components
significantly decreased compared to the Ach-induced oscillations (1.0 ± 0.5 vs. 29.3 ± 2.5% and 7.5 ± 1.0 vs. 23 ±
2%, respectively; p < 0.01, ANOVA, and p < 0.05, Bonferroni’s post hoc test). Therefore, the treatment abolished
the ULF component, while the effect on VLF was partial.
The LF, HF and VHF components showed a significant
increase in their percent power compared to Ach-triggered oscillatory components (42 ± 2 vs. 24.0 ± 1.7%; 7.0
± 1.5 vs. 2.0 ± 0.5%; and 24.5 ± 1.8 vs. 3.0 ± 1.1%, respectively; p < 0.01, ANOVA, and p < 0.05, Bonferroni’s post
hoc test), while the ILF component did not significantly
change (Fig. 8). Finally, in Figure 9 we report the main
frequency components in order 2 arterioles after the different treatments.
Color version available online
23
Diameter, μm
22
21
20
19
18
17
a
0
5
10
15
Time, min
20
25
30
0.10
0.06
0.04
0.02
pressed as percent normalized power spectral density (PSD: μm2/Hz) in a rat treated
with Nω-nitro-L-arginine prior to acetylcholine. cpm, cycles per minute. L-NIO
caused a decrease in VLF spectral density.
p < 0.01 vs. baseline conditions.
0
b
1
10
Frequency, cpm
100
200
300
Color version available online
Fig. 6. Rhythmic diameter changes in an
order 2 arteriole (a) and the main corresponding frequency components (b), ex-
PSD, μm2/cpm
0.08
19.0
Diameter, μm
18.5
18.0
17.5
17.0
16.5
16.0
a
0
5
10
15
Time, min
20
25
30
0.10
pressed as percent normalized power spectral density (PSD: μm2/Hz) in a rat treated
with charybdotoxin plus apamin. cpm, cycles per minute. Charybdotoxin plus apamin caused a decrease in the spectral density of the ultra-low-frequency component.
p < 0.01 vs. baseline conditions.
Cerebral Vasomotion and LF Oscillations
0.06
0.04
0.02
0
b
1
10
Frequency, cpm
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100
200
300
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Fig. 7. Rhythmic diameter changes in an
order 2 arteriole (a) and the main corresponding frequency components (b), ex-
PSD, μm2/cpm
0.08
Color version available online
31
Diameter, μm
30
29
28
27
26
a
0
5
10
15
Time, min
20
25
30
0.10
0.06
0.04
0.02
0
b
component). Higher-frequency components were in the
ranges of 0.2–2.0 Hz (12–120 cpm, HF component) and
2.5–4.5 Hz (150–270 cpm, VHF component). In all orders
of arterioles we found the same frequency components;
therefore, it is conceivable to suggest that the mechanisms
modulating arteriolar diameter changes appear to be effective in all vessels of the microvascular networks. The
decrease in the spectral density of the ILF and HF components in order 4 arterioles may be related to the higher
response of these arterioles to the factors modulating the
ULF and VLF components. Previous data indicate that
30-min recordings are adequate for evaluation of VLF
components, including ULF ones, suggested to be linked
to NO-independent endothelial activity [22]. However,
we performed several preliminary evaluations of experimental data to estimate the ULF component. The present
data demonstrate that our evaluation of the ULF component was consistent. Previously, we demonstrated that oscillations in diameter are transferred along the arteriolar
networks [37].
Our data are in agreement with previous results, indicating that oscillations in blood flow are related to frequency components modulated by several mechanisms
354
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1
10
Frequency, cpm
100
200
300
[32]. In particular, the data, derived from human skin via
the LDPM technique, show that the LF components in
blood flow oscillations are related to endothelial cell activity (range: 0.0095–0.02 Hz/0.57–1.2 cpm), sympathetic
nervous system discharge (range: 0.02–0.06 Hz/1.2–3.6
cpm), and myogenic mechanism (range: 0.06–0.2 Hz/3.6–
12 cpm). It was suggested that the frequency component
in the range of 0.0095–0.02 Hz (0.57–1.2 cpm) may be
related to NO release by endothelial cells [14]. We observed that arteriolar diameter oscillations were due to
the properties of arteriolar smooth muscle cells (range
0.06–0.2 Hz/3.6–12 cpm) and respiratory and heart rates
[5, 37, 38]. It is interesting to note that the present results
were derived from anesthetized animals, while our previous observations were carried out in awake animals. Anesthesia is known to cause arteriolar dilation and a decrease in the spectral density of several frequency components. However, our results demonstrated that chloralose
anesthesia does not abolish arteriolar diameter changes,
even though it may reduce the spectral density of several
frequency components [39].
To clarify the origin of the mechanisms involved in the
modulation of arteriolar diameter oscillations, we used
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pressed as percent normalized power spectral density (PSD: μm2/Hz) in a rat treated
with charybdotoxin plus apamin prior to
acetylcholine. cpm, cycles per minute.
Charybdotoxin plus apamin prior to acetylcholine caused a decrease in the spectral
density of the ULD component. p < 0.01 vs.
baseline conditions.
PSD, μm2/cpm
0.08
Fig. 8. Rhythmic diameter changes in an
order 2 arteriole (a) and the main corresponding frequency components (b), ex-
100
100
90
90
80
70
60
50
40
30
30
10
0
0
ULF
VLF
ILF
LF
HF
VHF
b
70
Normalized PSD, %
80
70
60
50
¹
*°
40
10
0
ILF
¹
*°
ULF
¹
*°
VLF
LF
*°
*°
HF
VHF
*°
LF
HF
VHF
d
30
0
100
90
80
80
70
70
60
¹
*°
50
40
¹
30
*°
*°
20
¹
*°
ULF
*°
¹
VLF
ILF
LF
HF
¹
ULF
f
*°
VLF
*°
ILF
LF
HF
VHF
¹
40
¹
30
0
¹
*°
50
10
VHF
¹
60
¹
¹
*°
*°
ULF
VLF
ILF
20
¹
*°
*°
40
10
¹
¹
20
¹
ILF
50
90
0
VLF
*°
60
100
10
ULF
*°
90
80
30
*°
100
Normalized PSD, %
Normalized PSD, %
40
10
20
Normalized PSD, %
50
20
90
e
60
20
100
c
70
*°
*°
¹
*°
LF
HF
VHF
Fig. 9. Comparison between the main frequency component
changes expressed as percent normalized power spectral density
(PSD) in order 2 arterioles in: a sham-operated rat (a), a rat treated with acetylcholine (b), a rat treated with papaverine (c), a rat
treated with Nω-nitro-L-arginine prior to acetylcholine (d), a rat
treated with charybdotoxin plus apamin (e), and a rat treated with
charybdotoxin plus apamin prior to acetylcholine (f). cpm, cycles
per minute; ULF, ultra-low frequency; VLF, very low frequency;
ILF, intermediate frequency; LF, low frequency; HF, high frequency; VHF, very high frequency (n = 11 arterioles for each group).
* p < 0.01 vs. baseline; ° p < 0.01 vs. the sham-operated group;
◇ p < 0.01 vs. the acetylcholine-treated group.
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a
Normalized PSD, %
Normalized PSD, %
80
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dependent frequency component in human cutaneous
blood flow oscillations was in the range of 0.0095–0.02 Hz
(0.57–1.2 cpm, VLF) [14].
The main purpose of the present study was to clarify
the mechanism modulating the frequency components in
the pial arteriolar oscillations. In particular, the ULF
component present in the human cutaneous blood flow
oscillations has been defined as NO independent. The
same component was detected in the oscillations of pial
arteriolar diameter, representing an average of 5–8% of
the total spectral density. Several authors have suggested
that EDHF is a powerful vasodilator influencing small resistance arteries and, consequently, an important modulator of blood pressure and flow [40]. EDHF is activated
by an increase in endothelial [Ca2+]i, which stimulates
2 Ca2+-sensitive K channels, i.e., SKCa and IKCa; consequently, apamin and charybdotoxin applied in combination blocked EDHF responses. Our data indicate that the
local application of charybdotoxin plus apamin, suggested EDHF inhibitors, significantly increased the total
spectral density of diameter oscillations. However, they
reduced the relative spectral density of the ULF component before Ach administration. After Ach injection, charybdotoxin plus apamin blunted the ULF component;
moreover, they affected the VLF component, which was
significantly decreased compared to Ach-induced changes. Therefore, the main target of charybdotoxin plus apamin was the ULF component, but the VLF component
was also affected. These responses demonstrated a clear
relationship between the release of the suggested EDHF
and ULF components in our experimental model, while
the VLF component was less involved.
It is worth noting that local administration of INDO
was unable to influence the VLF component, showing
that prostaglandins did not participate in the modulation
of this component compared to the suggested EDHF,
able to hyperpolarize smooth muscle cells in the arteriolar
wall and to dilate arterioles. This is the first demonstration of EDHF involvement in the regulation of pial arteriolar diameter oscillations in an experimental model.
Local administration of Ach and the other substances
were able to influence the other frequency components,
because those related to sympathetic nervous system discharge (ILF), myogenic activity (LF), respiration (HF),
and heart rate (VHF) changed according to the increase
or decrease in the relative spectral density of all of the
other components. However, taking into account our
present data, it is possible to suggest that arteriolar vasodilation is accompanied by an increase in spectral density
of VLF components, while the HF and VHF frequency
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local administration of Ach on pial arteriolar networks.
Our data show that topical Ach induced an increase in the
total power spectral density, indicating an increase in the
power of oscillations. Therefore, all components presented an increase, but it was important to know the spectral
density distribution among the frequency components.
Consequently, we reported the relative spectral density to
compare the power of all of the components. Therefore,
we observed that the spectral density of the ULF and VLF
components significantly increased compared to the values of sham-operated animals (Fig. 9a). The HF and VHF
components significantly decreased when comparing the
relative powers (Fig. 9a). For comparison of Ach-induced
dilation, known to be caused by an NO-dependent mechanism, we tested the effects of Pap administration, an
NO-independent vasodilator. Pap induced an increase in
the power of oscillations; in particular, the relative spectral density of the ULF and VLF components significantly increased, while the ILF and LF components did not
significantly change and the HF and VHF components
decreased compared to baseline conditions (Fig. 9c).
However, Pap-induced increases in the relative spectral
density of the ULF and VLF components were lower
compared to those due to Ach treatment (13.7 ± 1.2 vs.
29.3 ± 2.5% and 13.5 ± 0.9 vs. 23.0 ± 2.0%, respectively;
p < 0.01). Moreover, the HF and VHF components did
not decrease to the same extent as observed with Ach administration (6.2 ± 0.8 vs. 2.0 ± 0.5% and 32.2 ± 3.5 vs. 3.0
± 1.1%, respectively; p < 0.01; Fig. 5). Therefore, Ach was
able to significantly reduce the HF and VHF components
compared to baseline conditions and Pap-induced changes (Fig. 9b).
According to these results, it is possible to hypothesize
that the mechanism regulating the component in the
range of 0.0095–0.02 Hz was predominantly NO dependent. In particular, NO is known to interfere with vascular smooth muscle cell contraction, inhibiting the higherfrequency components. As a consequence of this mechanism, lower-frequency components were detected and
evaluated by power spectrum analysis. The ULF and VLF
components presented higher spectral densities compared to those present under baseline conditions. Moreover, the administration of L-NNA, an eNOS inhibitor,
significantly increased the total spectral density of diameter oscillations; however, there was a significant reduction in the relative spectral density of the frequency component in the range of 0.0095–0.02 Hz (0.57–1.2 cpm,
VLF). These data further support the NO-dependent origin of this frequency component. Our results are in agreement with previous observations indicating that the NO-
components are markedly reduced. The same patterns
have been demonstrated in both Ach- and Pap-induced
dilation, even though with Pap there was a higher spectral
density of the VHF component. Arteriolar constriction,
on the other hand, was characterized by a decrease in the
spectral density of VLF components, while the LF and
VHF components revealed a higher spectral density. The
changes in the relative spectral density of each component were strictly related to the variations in the relative
spectral density of the other frequency components. Finally, no influence of locally applied substances on respiratory rate or heart rate was observed.
Therefore, our observations support the hypothesis
that substances released by endothelial cells may contribute to the regulation of pial arteriolar diameter changes
useful to modulate distribution of blood flow in cerebral
circulation. This mechanism cooperates with NO release
to control arteriolar tone and capillary perfusion, assuring adequate blood flow to neurons in the different brain
regions. These substances may be similar or identical to
EDHF, able to affect the rhythmic changes in arteriolar
diameter.
Undoubtedly, we cannot exclude that both endothelium-derived factors may be affected by L-NNA or charybdotoxin plus apamin administration. We would like to
point out that in our model the effects of L-NNA appear
to be greater on the NO-related frequency component
(VLF), while charybdotoxin plus apamin appear to be
more effective on the other related component (like
EDHF), especially after Ach administration.
In conclusion, the regulation of the blood flow distribution is due to several mechanisms; one of these appears
to be related to a substance released by the endothelial
cells modulating the vascular tone. This endothelial-derived factor contributes to the overall modulation of arteriolar tone and arteriolar responses, such as dilation under different physiological or pathophysiological conditions, facilitating perfusion of cerebral tissue according to
its requirements. This factor exerts its effect on arteriolar
smooth muscle cells endowed in the arteriolar walls, able
to constrict and dilate. These cells represent the key target, they motor activity of which may be affected by several effectors, resulting in modulation of the blood flow
distribution in the cerebral circulation by regulation of
the arteriolar diameter.
Disclosure Statement
The authors have no conflicts of interest to disclose.
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